The present invention relates to the use of subtilase variants for removal of egg stains from laundry or from hard surfaces. In particular the present invention relates to the use of a subtilase variant for removal of egg stains from laundry or from hard surfaces, where the subtilase variant comprises at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering, vide infra). These subtilase variants are useful exhibiting excellent or improved wash performance on egg stains when used in e.g. cleaning or detergent compositions, such as laundry detergent compositions and dishwash composition, including automatic dishwash compositions. The present invention also relates to novel subtilase variants, to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention. Further, the present invention relates to cleaning and detergent compositions comprising the variants of the invention.
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.
An increasing number of commercially used proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM(copyright) (Novo Nordisk A/S), RELASE(copyright) (Novo Nordisk A/S), MAXAPEM(copyright) (Gist-Brocades N.V.), PURAFECT(copyright) (Genencor International, Inc.).
Further, a number of protease variants are described in the art. A thorough list of prior art protease variants is given in WO 99/27082.
However, even though a number of useful protease variants have been described, there is still a need for new improved proteases or protease variants for a number of industrial uses.
In particular, the problem of removing egg stains from e.g. laundry or hard surfaces has been pronounced due to the fact that many proteases are inhibited by substances present in the egg white. Examples of such substances include trypsin inhibitor type IV-0 (Ovo-inhibitor) and trypsin inhibitor type III-0 (Ovomucoid).
Therefore, an object of the present invention, is to provide improved subtilase variants, which are not, or which are only to a limited extent, inhibited by such substances. A further object of the present invention is to provide improved subtilase variants, which are suitable for removal of egg stains from, for example, laundry and/or hard surfaces.
Thus, in a first aspect the present invention relates to the use of a subtilase variant for removal of egg stains from laundry or from hard surfaces, the subtilase variant comprising at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering).
In a second aspect the present invention relates to a subtilase variant selected from the group consisting of
a variant comprising at least one additional amino acid residue in the active site (b) loop corresponding to the insertion of at least one additional amino acid residue between positions 98 and 99 and further comprising at least one additional modification (BASBPN numbering), and
a variant comprising at least one additional amino acid residue in the active site (b) loop corresponding to the insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising at least one additional modification (BASBPN numbering),
where the variantxe2x80x94when tested in the xe2x80x9cOvo-inhibition Assayxe2x80x9d disclosed in Example 4 hereinxe2x80x94has a Residual Activity of at least 10%.
In a third aspect the present invention relates to a subtilase variant selected from the group consisting of
a variant comprising an insertion of at least one additional amino acid residue between positions 98 and 99 and further comprising a substitution in positions 133 and 143,
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising a substitution in position 99,
a variant comprising an insertion of at least one additional amino acid residue between positions 98 and 99 and further comprising substitutions in positions 167, 170 and 194,
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising an insertion of at least one additional amino acid residue between positions 216 and 217,
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising an insertion of at least one additional amino acid residue between positions 217 and 218,
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising an insertion of at least one additional amino acid residue between positions 42 and 43, and
a variant comprising an insertion of at least one additional amino acid residue between positions 99 and 100 and further comprising an insertion of at least one additional amino acid residue between positions 129 and 130.
In a fourth aspect the present invention relates to an isolated DNA sequence encoding a subtilase variant of the invention.
In a fifth aspect the present invention relates to an expression vector comprising the isolated DNA sequence of the invention.
In a sixth aspect the present invention relates to a microbial host cell transformed with the expression vector of the invention.
In a seventh aspect the present invention relates to a method for producing a subtilase variant according to the invention, wherein a host according to the invention is cultured under conditions conducive to the expression and secretion of said variant, and the variant is recovered.
In an eight aspect the present invention relates to a cleaning or detergent composition, preferably a laundry or dishwash composition, comprising the variant of the invention.
In a ninth aspect the present invention relates to a method for removal of egg stains from a hard surface or from laundry, the method comprising contacting the egg stain-containing hard surface or the egg stain-containing laundry with a cleaning or detergent composition, preferably a laundry or dishwash composition, containing a subtilase variant comprising at least one additional amino acid residue in the active site loop (b) region from position 95 to 103 (BASBPN numbering).
Still other aspect of the present invention will be apparent from the below description and from the appended claims.
Concerning alignment and numbering reference is made to FIG. 1 which shows an alignments between subtilisin BPN"" (a) (BASBPN) and subtilisin 309 (BLSAVI) (b).
These alignments are in this patent application used as a reference for numbering the residues.
Definitions
Prior to discussing this invention in further detail, the following terms and conventions will first be defined.
Nomenclature and Conventions for Designation of Variants
In describing the various subtilase enzyme variants produced or contemplated according to the invention, the following nomenclatures and conventions have been adapted for ease of reference:
A frame of reference is first defined by aligning the isolated or parent enzyme with subtilisin BPN"" (BASBPN).
The alignment can be obtained by the GAP routine of the GCG package version 9.1 to number the variants using the following parameters: gap creation penalty=8 and gap extension penalty=8 and all other parameters kept at their default values.
Another method is to use known recognized alignments between subtilases, such as the alignment indicated in WO 91/00345. In most cases the differences will not be of any importance.
Thereby a number of deletions and insertions will be defined in relation to BASBPN. In FIG. 1, subtilisin 309 (Savinase(copyright)) has 6 deletions in positions 36, 58, 158, 162, 163, and 164 in comparison to BASBPN. These deletions are in FIG. 1 indicated by asterixes (*).
The various modifications performed in a parent enzyme is indicated in general using three elements as follows:
Original Amino Acid Position Substituted Amino Acid
The notation G195E thus means a substitution of a glycine in position 195 with a glutamic acid.
In the case where the original amino acid residue may be any amino acid residue, a short hand notation may at times be used indicating only the position and substituted amino acid:
Position Substituted Amino Acid
Such a notation is particular relevant in connection with modification(s) in homologous subtilases (vide infra).
Similarly when the identity of the substituting amino acid residue(s) is immaterial:
Original Amino Acid Position
When both the original amino acid(s) and substituted amino acid(s) may comprise any amino acid, then only the position is indicated, e.g.: 170.
When the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), then the selected amino acids are indicated inside brackets:
Original Amino Acid Position {Substituted Amino Acid1, . . . , Substituted Amino Acidn}
For specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue.
Substitutions
The substitution of glutamic acid for glycine in position 195 is designated as:
Gly195Glu or G195E
or the substitution of any amino acid residue acid for glycine in position 195 is designated as:
Gly195Xaa or G195X
or
Gly195 or G195
The substitution of serine for any amino acid residue in position 170 would thus be designated
Xaa170Ser or X170S.
or
170Ser or 170S
Such a notation is particular relevant in connection with modification(s) in homologous subtilases (vide infra). 170Ser is thus meant to comprise e.g. both a Lys170Ser modification in BASBPN and Arg170Ser modification in BLSAVI (cf. FIG. 1).
For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of glycine, alanine, serine or threonine for arginine in position 170 would be indicated by
Arg170{Gly,Ala,Ser,Thr} or R170{G,A,S,T}
to indicate the variants
R170G, R170A, R170S, and R170T.
Deletions
A deletion of glycine in position 195 will be indicated by:
Gly195* or G195*
Correspondingly the deletion of more than one amino acid residue, such as the deletion of glycine and leucine in positions 195 and 196 will be designated
Gly195*+Leu196* or G195*+L196*
Insertions
The insertion of an additional amino acid residue such as e.g. a lysine after G195 is indicated by:
Gly195GlyLys or G195GK;
or, when more than one amino acid residue is inserted, such as e.g. a Lys, Ala and Ser after G195 this will be indicated as:
Gly195GlyLysAlaSer or G195GKAS (SEQ ID NO:1)
In such cases the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example the sequences 194 to 196 would thus be:
In cases where an amino acid residue identical to the existing amino acid residue is inserted it is clear that a degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G195GG. The same actual change could just as well be indicated as A194AG for the change from
Such instances will be apparent to the skilled person, and the indication G195GG and corresponding indications for this type of insertions are thus meant to comprise such equivalent degenerate indications.
Filling a Gap
Where a deletion in an enzyme exists in the reference comparison with the subtilisin BPN"" sequence used for the numbering, an insertion in such a position is indicated as:
*36Asp or *36D
for the insertion of an aspartic acid in position 36
Multiple Modifications
Variants comprising multiple modifications are separated by pluses, e.g.:
Arg170Tyr+Gly195Glu or R170Y+G195E
representing modifications in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.
Thus, Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr} designates the following variants:
This nomenclature is particular relevant relating to modifications aimed at substituting, replacing, inserting or deleting amino acid residues having specific common properties, such as residues of positive charge (K, R, H), negative charge (D, E), or conservative amino acid modification(s) of e.g. Tyr167{Gly,Ala,Ser,Thr}+Arg170{Gly,Ala,Ser,Thr}, which signifies substituting a small amino acid for another small amino acid. See section xe2x80x9cDetailed description of the inventionxe2x80x9d for further details.
Proteases
Enzymes cleaving the amide linkages in protein substrates are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3).
Numbering of Amino Acid Positions/residues
If nothing else is mentioned the amino acid numbering used herein correspond to that of the subtilase BPN"" (BASBPN) sequence. For further description of the BPN"" sequence, see FIG. 1 or Siezen et al., Protein Engng. 4 (1991) 719-737.
Serine Proteases
A serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 xe2x80x9cPrinciples of Biochemistry,xe2x80x9d Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
The bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
Subtilases
A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997).
One subgroup of the subtilases, I-S1 or xe2x80x9ctruexe2x80x9d subtilisins, comprises the xe2x80x9cclassicalxe2x80x9d subtilisins, such as subtilisin 168 (BSS168), subtilisin BPN"" (SEQ ID NO:38), subtilisin Carlsberg (ALCALASE(copyright), NOVO NORDISK A/S), and subtilisin DY (BSSDY).
A further subgroup of the subtilases, I-S2 or high alkaline subtilisins, is recognized by Siezen et al. (supra). Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (MAXACAL(copyright), Gist-Brocades NV), subtilisin 309 (SEQ ID NO:49) (SAVINASE(copyright), NOVO NORDISK A/S), subtilisin 147 (BLS147) (ESPERASE(copyright), NOVO NORDISK A/S), and alkaline elastase YaB (BSEYAB).
Savinase(copyright)
SAVINASE(copyright) is marketed by NOVO NORDISK A/S. It is subtilisin 309 from B. Lentus and differs from BAALKP only in one position (N87S, see FIG. 1 herein). SAVINASE(copyright) has the amino acid sequence designated b) in FIG. 1 and as shown in SEQ ID NO:49.
Parent Subtilase
The term xe2x80x9cparent subtilasexe2x80x9d describes a subtilase defined according to Siezen et al. (1991 and 1997). For further details see description of xe2x80x9cSUBTILASESxe2x80x9d immediately above. A parent subtilase may also be a subtilase isolated from a natural source, wherein subsequent modifications have been made while retaining the characteristic of a subtilase. Furthermore, a parent subtilase may also be a subtilase which has been prepared by the DNA shuffling technique, such as described by J. E. Ness et al., Nature Biotechnology, 17, 893-896 (1999). Alternatively the term xe2x80x9cparent subtilasexe2x80x9d may be termed xe2x80x9cwild type subtilasexe2x80x9d.
Modification(s) of a Subtilase Variant
The term xe2x80x9cmodification(s)xe2x80x9d used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase. The modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
Subtilase Variant
In the context of this invention, the term subtilase variant or mutated subtilase means a subtilase that has been produced by an organism which is expressing a mutant gene derived from a parent microorganism which possessed an original or parent gene and which produced a corresponding parent enzyme, the parent gene having been mutated in order to produce the mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.
Homologous Subtilase Sequences
Specific active site loop regions, and amino acid insertions in said loops of subtilisin 309 are identified for modification herein to obtain a subtilase variant of the invention.
However, the invention is not limited to modifications of this particular subtilase, but extend to other parent (wild-type) subtilases, which have a homologous primary structure to that of subtilisin 309. The homology between two amino acid sequences is in this context described by the parameter xe2x80x9cidentityxe2x80x9d.
In order to determine the degree of identity between two subtilases the GAP routine of the GCG package version 9.1 can be applied (infra) using the same settings. The output from the routine is besides the amino acid alignment the calculation of the xe2x80x9cPercent Identityxe2x80x9d between the two sequences.
Based on this description it is routine for a person skilled in the art to identify suitable homologous subtilases and corresponding homologous active site loop regions, which can be modified according to the invention.
Isolated DNA Sequence
The term xe2x80x9cisolatedxe2x80x9d, when applied to a DNA sequence molecule, denotes that the DNA sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5xe2x80x2 and 3xe2x80x2 untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985). The term xe2x80x9can isolated DNA sequencexe2x80x9d may alternatively be termed xe2x80x9ca cloned DNA sequencexe2x80x9d.
Isolated Protein
When applied to a protein, the term xe2x80x9cisolatedxe2x80x9d indicates that the protein has been removed from its native environment.
In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. xe2x80x9chomologous impuritiesxe2x80x9d (see below)).
An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE. Further it is preferred to provide the protein in a highly purified form, i.e., more than 40% pure, more than 60% pure, more than 80% pure, more preferably more than 95% pure, and most preferably more than 99% pure, as determined by SDS-PAGE.
The term xe2x80x9cisolated proteinxe2x80x9d may alternatively be termed xe2x80x9cpurified proteinxe2x80x9d.
Homologous Impurities
The term xe2x80x9chomologous impuritiesxe2x80x9d means any impurity (e.g. another polypeptide than the subtilase of the invention), which originate from the homologous cell where the subtilase of the invention is originally obtained from.
Obtained from
The term xe2x80x9cobtained fromxe2x80x9d as used herein in connection with a specific microbial source, means that the polynucleotide and/or subtilase produced by the specific source, or by a cell in which a gene from the source has been inserted.
Substrate
The term xe2x80x9csubstratexe2x80x9d used in connection with a substrate for a protease should be interpreted in its broadest form as comprising a compound containing at least one peptide bond susceptible to hydrolysis by a subtilisin protease.
Product
The term xe2x80x9cproductxe2x80x9d used in connection with a product derived from a protease enzymatic reaction should, in the context of the present invention, be interpreted to include the products of a hydrolysis reaction involving a subtilase protease. A product may be the substrate in a subsequent hydrolysis reaction.
Wash Performance
In the present context the term xe2x80x9cwash performancexe2x80x9d is used as an enzyme""s ability to remove egg stains present on the object to be cleaned during e.g. wash or hard surface cleaning. See also the xe2x80x9cModel Detergent Wash Performance Testxe2x80x9d in Example 3 herein.
Performance Factor
The term xe2x80x9cPerformance Factorxe2x80x9d is defined with respect to the below formula
P=Rvariantxe2x88x92Rparent
wherein P is the Performance Factor, Rvariant is the reflectance (measured at 460 nm) of the test material after being treated with a subtilase variant as described in the xe2x80x9cModel Detergent Wash Performance Testxe2x80x9d, and Rparent is the reflectance (measured at 460 nm) of the test material after being treated with the corresponding parent subtilase as described in the xe2x80x9cModel Detergent Wash Performance Testxe2x80x9d. For further details, see the xe2x80x9cModel Detergent Wash Performance Testxe2x80x9d in Example 3 herein.
Residual Activity
The term xe2x80x9cResidual Activityxe2x80x9d is defined as described in the xe2x80x9cOvo-inhibition Assayxe2x80x9d herein (see Example 4).